Under fasting conditions, increases in circulating glucagon stimulate hepatic glucose production via induction of the cAMP pathway. Conversely, increases in gut-derived glucagon-like peptide 1 (GLP1) during feeding enhance glucose clearance by promoting insulin release. The transcription factor CREB is thought to mediate long term effects of both peptide hormones, following its phosphorylation by PKA and association with CBP/P300. The transcriptional response to cAMP follows burst-attenuation kinetics; CREB activity peaks after 1 hour of stimulation, returning to baseline after 4-6 hours. In addition to their effects on CREB phosphorylation, glucagon and GLP1 also increase CREB activity by stimulating its association with the cAMP Regulated Transcriptional Coactivators (CRTCs/TORCs), latent cytoplasmic CREB cofactors that translocate to the nucleus following their dephosphorylation in response to cAMP. CRTC1 is expressed only in brain, while CRTC2 and CRTC3 are co-expressed in most tissues. The extent to which CRTC2 and CRTC3 function on overlapping or distinct subsets of CREB target genes is unclear, however. In the previous grant period, we showed that the CREB/CRTC2 pathway contributes importantly to fasting glucose production; acute depletion of CRTC2 in liver substantially lowers blood glucose concentrations and gluconeogenic gene expression, while over-expression of wild-type and to a greater extent phosphorylation-defective CRTC2 increases gluconeogenesis. By contrast with effects of acute hepatic CRTC2 knockdown, mice with a whole-body knockout of CRTC2 show only modest reductions in fasting glucose levels; and they develop an insulin secretion defect as they age. These results point to the involvement of additional CREB coactivators that compensate for loss of CRTC2 in liver, and they suggest that CRTC2 expression in pancreatic islets also modulates circulating glucose concentrations through its effects on insulin secretion. Supporting the latter, MafA, a beta cell transcription factor that is required for insulin secretion, is strongly upregulated by CREB and CRTC2. Proposed studies during the upcoming grant period focus on the hypothesis that members of the CRTC family exert overlapping effects on CREB activity. The importance of a newly identified CREB interacting protein in potentiating CREB activity and compensating for loss of CRTC2 in CRTC2 mutant mice will be tested. Finally the role of a potent CREB inhibitor, which is upregulated in pancreatic islets under hyperglycemic conditions, in promoting resistance to Gs-coupled receptor signaling, will be evaluated. Three aims are proposed; they extend the previous work by addressing the mechanisms by which the CREB pathway promotes gluconeogenesis in liver and facilitates insulin secretion from pancreatic islets. In Aim 1, we will use mice with floxed alleles of CRTC2 and CRTC3 to evaluate the relative roles of these coactivators in modulating hepatic gluconeogenesis and insulin secretion. We will generate mice with tissue specific knockouts of CRTC2 and CRTC3 in liver or pancreatic islets. Do CRTC2 and CRTC3 exert overlapping effects on gluconeogenic gene expression in liver? Do they promote insulin secretion by upregulating the leucine zipper factor MafA? In Aim 2, we will test the role of BRD2-a bromodomain protein identified in a proteomic screen for CREB associated proteins- in stimulating expression of gluconeogenic genes. We will characterize domains in BRD2 and CREB that mediate this interaction; and the role of CREB acetylation in modulating the BRD2:CREB association will also be tested. We will evaluate whether inhibition of BRD2, through administration of a selective bromodomain inhibitor, improves glucose levels in the setting of insulin resistance. In Aim 3, we will examine the mechanism by which CREB target gene expression in pancreatic islets is down-regulated in insulin resistance. In particular, we will investigate the role of Protein Kinase Inhibitor beta (PKIB) in interfering with GLP1 and other hormones, following its upregulation in response to hyperglycemia: PKIB knockout mice will be used to determine whether depletion of this inhibitor improves pancreatic islet function in the setting of insulin resistance. Taken together, the proposed studies will provide new insight into mechanisms by which glucagon and GLP1 promote glucose balance through their effects on the CREB pathway in liver and pancreatic beta cells.